All life forms require a ring-shaped sliding clamp to coordinate replication of their genome. These sliding clamps act as master regulators of DNA replication, coordinating replisome action with other cellular processes. These master regulators are themselves regulated by large ATPase machines called clamp loaders that either install or remove sliding clamps from DNA. This project seeks to gain an atomic-level understanding of clamp loader mechanism. These protein remodeling machines open the sliding clamp ring as a key step in their action. We have found that in the key intermediate complex?consisting of an open clamp, an ATP-bound clamp loader and the target DNA?the protein components form an open spiral that matches the helical symmetry of DNA. This symmetric spiral activates ATP hydrolysis leading to clamp closure and release of the loaded clamp. In Aim 1, we now turn our attention to the critical first two steps of the reaction: the opening of the clamp and the binding of DNA to the inner chamber of the complex. We will identify the conformational changes in the clamp loader complex that allow for opening the clamp ring, as well as how the assembly can rapidly bind a specific DNA structure in the tight confines of the complex's interior. In Aim 2, we investigate how the single subunit change in the clamp loader complex (Rfc1 replaced with Elg1) converts a dedicated clamp loader into a dedicated unloader. This work will not only reveal the mechanism and structure of a key protein involved in cancer development, but will also provide a blueprint for how an ATPase machine can be reprogrammed to perform a reverse reaction. Finally, in Aim 3 we explore how replacement of the Rfc1 subunit with the Ctf18 protein leads to an assembly that is bifunctional as both a loader and unloader, and that connects DNA replication to the process of sister chromatid cohesion. Our structures and analysis of this complex will reveal how an ATPase machine can be mechanistically flexible to catalyze both forward and reverse reactions. In addition, this work will provide insight into how this mysterious complex can link the seemingly disparate processes of DNA replication and sister chromatid cohesion. Because clamp loaders and sliding clamps are fundamental to all life, the structural insights that we obtain from completing our aims will be used for developing novel antimicrobial or chemotherapeutic drugs.